Hydrophobic modified pres-derived peptides of hepatitis B virus (HBV) and their use as vehicles for the specific delivery of compounds to the liver

ABSTRACT

The present invention relates to hydrophobic modified preS-derived peptides of hepatitis B virus (HBV) which are versatile vehicles for the specific delivery of compounds to the liver, preferably to hepatocytes, in vitro as well as in vivo. Any kind of compound, but in particular drugs, such as interferons, viral reverse transcriptase inhibitors or core assembly inhibitors, and/or labels can be specifically targeted to the liver and so be enriched in the liver. This liver targeting can further be used for the targeted diagnosis, prevention and/or treatment of liver diseases or disorders, such as hepatitis, malaria, hepatocellular carcinoma (HCC), as well as for the prevention of HAV, HBV, HCV and/or HDV infection. The present invention relates to pharmaceutical compositions comprising said hydrophobic modified preS-derived peptide(s) and the compound(s) to be specifically delivered to the liver. The present invention furthermore relates to a method for the combined treatment of a liver disease and the prevention of HAV, HBV, HCV and/or HDV infection. The present invention relates also to the use of the preS-derived peptides in gene therapy and the delivery of immunogenic epitopes for hepatocyte-mediated antigen presentation to activate liver-directed immunological responses.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a National Stage Application of InternationalApplication Number PCT/EP2009/000477, filed Jan. 26, 2009; which claimsthe benefit of U.S. Provisional Application Ser. No. 61/062,347, filedJan. 25, 2008; which are incorporated herein by reference in theirentirety.

The present invention relates to hydrophobic modified preS-derivedpeptides of hepatitis B virus (HBV) which are versatile vehicles for thespecific delivery of compounds to the liver, preferably to hepatocytes,in vitro as well as in vivo. Any kind of compound, but in particulardrugs, such as interferons, viral reverse transcriptase inhibitors orcore assembly inhibitors, and/or labels can be specifically targeted tothe liver and so be enriched in the liver. This liver targeting canfurther be used for the targeted diagnosis, prevention and/or treatmentof liver diseases or disorders, such as hepatitis, malaria,hepatocellular carcinoma (HCC), as well as for the prevention of HAV,HBV, HCV and/or HDV infection. The present invention relates topharmaceutical compositions comprising said hydrophobic modifiedpreS-derived peptide(s) and the compound(s) to be specifically deliveredto the liver. The present invention furthermore relates to a method forthe combined treatment of a liver disease and the prevention of HAV,HBV, HCV and/or HDV infection. The present invention relates also to theuse of the preS-derived peptides in gene therapy and the delivery ofimmunogenic epitopes for hepatocyte-mediated antigen presentation toactivate liver-directed immunological responses.

BACKGROUND OF THE INVENTION

The liver, an organ which is present in vertebrates and other animals,plays a major role in the metabolism and has a number of functions inthe body, including glycogen storage, decomposition of red blood cells,synthesis of plasma proteins, and detoxification. The liver also is thelargest gland in the human body. It lies below the diaphragm in thethoracic region of the abdomen. It produces bile, an alkaline compoundwhich aids in digestion, via the emulsification of lipids. It alsoperforms and regulates a wide variety of high-volume biochemicalreactions requiring specialized tissues.

Hepatocytes make up 70 to 80% of the cytoplasmic mass of the liver.Hepatocytes are involved in protein synthesis, protein storage andtransformation of carbohydrates, synthesis of cholesterol, bile saltsand phospholipids, and detoxification, modification and excretion ofexogenous and endogenous substances. The hepatocyte also initiates theformation and secretion of bile.

There is a wide number of known liver diseases, such as:

-   -   Hepatitis: inflammation of the liver, caused mainly by various        viruses but also by certain poisons, autoimmunity or hereditary        conditions;    -   Cirrhosis: the formation of fibrous tissue in the liver,        replacing dead liver cells. The death of the liver cells can for        example be caused by viral hepatitis, alcoholism or contact with        other liver-toxic chemicals;    -   Haemochromatosis: a hereditary disease causing the accumulation        of iron in the body, eventually leading to liver damage;    -   Cancer of the liver: primary hepatocellular carcinoma (HCC) or        cholangiocarcinoma and metastatic cancers, usually from other        parts of the gastrointestinal tract;    -   Wilson's disease: a hereditary disease which causes the body to        retain copper;    -   Primary sclerosing cholangitis: an inflammatory disease of the        bile duct, autoimmune in nature;    -   Primary biliary cirrhosis: autoimmune disease of small bile        ducts;    -   Budd-Chiari syndrome: obstruction of the hepatic vein;    -   Gilbert's syndrome: a genetic disorder of bilirubin metabolism,        found in about 5% of the population;    -   Glycogen storage disease type II: the build-up of glycogen        causes progressive muscle weakness (myopathy) throughout the        body and affects various body tissues, particularly in the        heart, skeletal muscles, liver and nervous system.

There are also many pediatric liver disease, such as biliary atresia,alpha-1-antitrypsin deficiency, alagille syndrome, and progressivefamilial intrahepatic cholestasis; as well as metabolic diseases.

Furthermore, several pathogens and parasites, especially of tropicaldiseases, have a liver stage during their life cycle.

For instance malaria, which is one of the most common infectiousdiseases and an enormous public-health problem. Malaria is caused byprotozoan parasites of the genus Plasmodium.

The most serious forms of the disease are caused by Plasmodiumfalciparum and Plasmodium vivax, but other related species (Plasmodiumovale, Plasmodium malariae, and sometimes Plasmodium knowlesi) can alsoinfect humans. This group of human-pathogenic Plasmodium species isusually referred to as malaria parasites. Malaria parasites aretransmitted by female Anopheles mosquitoes. Malaria in humans developsvia two phases: an exoerythrocytic (hepatic phase or “liver stage”) andan erythrocytic phase. When an infected mosquito pierces a person's skinto take a blood meal, sporozoites in the mosquito's saliva enter thebloodstream and migrate to the liver. Within about 30 minutes of beingintroduced into the human host, they infect hepatocytes, multiplyingasexually and asymptomatically for a period of 6-15 days. Once in theliver these organisms differentiate to yield thousands of merozoiteswhich, following rupture of their host cells, escape into the blood andinfect red blood cells, thus beginning the erythrocytic stage of thelife cycle. The parasite escapes from the liver undetected by wrappingitself in the cell membrane of the infected host liver cell. Theparasite is relatively protected from attack by the body's immune systembecause for most of its human life cycle it resides within the liver andblood cells and is relatively invisible to immune surveillance. There isincreasing interest in developing drugs that specifically address theliver specific stages of malaria parasites (e.g. primaquin) in order toobviate the development of blood stages.

Another example is schistosomiasis or bilharziosis, which is a parasiticdisease caused by several species of flatworm. Although it has a lowmortality rate, schistosomiasis can be very debilitating. It is an oftenchronic illness that results from infection of the blood with aparasitic flatworm (schistosome). It causes debilitation and causesliver and intestinal damage. It is most commonly found in Asia, Africa,and South America, especially in areas with water that is contaminatedwith fresh water snails, which contain the parasite.

Hepatitis B virus (HBV), the cause for hepatitis B, is the prototype ofa family of small, enveloped DNA viruses of mammals and birds (1). TheHBV envelope encloses three proteins termed L—(large), M—(middle) andS—(small) (FIG. 1A). They share the C-terminal S-domain with fourtransmembrane regions. The M- and L-protein carry additional N-terminalextensions of 55 and, genotype-dependent, 107 or 118 aa (preS2- andpreS1) (FIG. 1B). In virions the stoichiometric ratio of L, M and S isabout 1:1:4, while the more abundantly secreted non-infectious subviralparticles (SVPs) contain almost exclusively S- and only traces ofL-protein (2). During synthesis, the preS1-domain of L is myristoylatedand at some point of the HBV life cycle translocated through theER-derived membrane. This modification is essential for infectivity(3,4). HBV as well as the other hepatotropic viruses have a livertropism, i.e. the liver is the tissue which specifically supports thegrowth of HBV.

The specific targeting of drugs aims at improving the efficacy oftherapies. In 1906 Paul Ehrlich introduced the expression “magic bullet”for the search of optimized treatment strategies. Since then, researchin the sense of drug targeting has focused on the development of carriersystems that increase the therapeutic concentration of a drug in targettissue such as tumors or pathogens and thus lowers the side effects forthe organism as a whole. Ideally, drug targeting should fulfill thefollowing criteria: 1) exclusive transfer of the drug to the requiredsite of action; 2) a minimum of effects for the remaining organism; 3)use of a pharmacologically inactive vector.

In order to carry a drug to a specific tissue different strategies arepursued. These are for example the use of prodrugs, from which thepharmacologically active part is released in the target tissue bytissue-specific enzymes. A further possibility is to couple effective,non-tissue-specific drugs to tissue-specific, but pharmacologicallyinert carrier systems like receptor-affine peptides or colloidalparticles.

Various drug carriers have been used to enhance liver targeting ofdrugs. A straightforward approach is based on the active phagocytosis ofthe reticuloendothelial system in the liver by delivering drugs inparticular carriers, such as liposomes and microspheres. For example, ithas been shown that following i.v. injection, particulate carriersincorporating a drug are mainly captured by the reticuloendothelialsystem in the liver, resulting in drug targeting of the liver (5). Onthe other hand, liver targeting of drugs with positively charged,water-soluble polymers is based on free extravasation of mostwater-soluble substances from the vascular system of the liver as wellas on negative charges on the liver cell surface (6). Thus, polymershave been used as the carrier to allow drugs to target to the liverbased on such anatomical and biochemical characteristics of the liver.More specific drug targeting of the liver has been attempted by usingasialoglycoprotein receptors of liver cells. The asialoglycoproteinreceptor (galactose receptor) is present on hepatocytes with highdensity. In addition, once a ligand binds to the galactose receptor, theligand-receptor complex is internalized which allows the cellular uptakeof galactosylated ligands (7).

U.S. Pat. No. 7,001,760 B2 disclose recombinant vectors derived fromhepatitis B virus (HBV), which can be used for gene therapy, such as thedelivery of therapeutic genes to liver cells and the expression ofheterologous genes in liver cells. However, HBV vectors have a limitedcapacity for packaging genes.

However, so far there exist no universal diagnostic, therapeutic and/orpreventative approaches that specifically target the organ liver in ananimal or bird, in particular a human, and, at the same time,specifically deliver a desired compound or drug to the liver.

Thus, there is the need for universal diagnostic, therapeutic and/orpreventative approaches that in a subject specifically target the organliver and specifically deliver a desired compound or drug to the liver

Furthermore, there exist no universal diagnostic, therapeutic and/orpreventative approaches that in a subject specifically target the organliver and specifically deliver a desired compound or drug to the liveras well as, at the same time, prevent entry of Hepatitis B and HepatitisD viruses into hepatocytes

Furthermore there is the need for the specific targeting of the liverwith antigenic epitopes to trigger the immune system, such as forvaccination.

The present invention, thus, aims to provide means and methods for theeffective, specific targeted diagnosis, prevention and/or treatment ofliver diseases and the regulation of the liver function.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by providing ahydrophobic modified preS-derived peptide of HBV.

Said hydrophobic modified preS-derived peptide has the general formula:[H_(m)-P-R_(n)]A_(o).

P is said preS-derived peptide and comprises the amino acid sequence ofthe “HBV consensus sequence” or variants thereof.

H is said hydrophobic modification of the preS-derived peptide P, whichis N-terminal of P and selected from acylation and addition ofhydrophobic moieties; wherein m is at least 1.

R is an optional C-terminal modification (i.e. n is 0 or at least 1) ofsaid preS-derived peptide P.

A is an optional anchor group (i.e. o is 0 or at least 1), which issuitable for the covalent attachment of the compound(s) and is linked toH, P and/or R.

According to the present invention this object is furthermore solved byproviding a pharmaceutical composition comprising at least onehydrophobic modified preS-derived peptide of HBV as defined herein andat least a compound to be specifically delivered to the liver,preferably to hepatocytes, as defined herein and optionally apharmaceutically acceptable carrier and/or excipient.

According to the present invention this object is furthermore solved byusing the hydrophobic modified preS-derived peptide(s) of HBV as vehicleor shuttle for the specific delivery of compound(s) to the liver.

According to the present invention this object is furthermore solved byproviding the hydrophobic modified preS-derived peptide(s) of HBV or thepharmaceutical composition(s) of the invention for the diagnosis,prevention and/or treatment of a liver disease or disorder or for theregulation of liver function.

According to the present invention this object is furthermore solved byusing the hydrophobic modified preS-derived peptide(s) of HBV or thepharmaceutical composition(s) of the invention for the manufacture of amedicament for the diagnosis, prevention and/or treatment of a liverdisease or disorder.

According to the present invention this object is furthermore solved bymethods for diagnosis, prevention and/or treatment of a liver disease ordisorder by utilizing the hydrophobic modified preS-derived peptide(s)of HBV or the pharmaceutical composition(s) of the invention.

According to the present invention this object is furthermore solved byproviding a method for the combined diagnosis, prevention and/ortreatment of a liver disease or disorder and the prevention of HBVand/or HDV infection by administering to a subject a conjugate asdefined herein, which comprises an hydrophobic modified preS-derivedpeptide of HBV and a compound, or a pharmaceutical composition asdefined herein.

According to the present invention this object is furthermore solved byproviding the use of a viral vector comprising a nucleic acid encoding apreS-derived peptide P as defined herein for the gene therapy of a liverdisease or disorder.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is tobe understood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. For the purpose of thepresent invention, all references cited herein are incorporated byreference in their entireties.

Hydrophobic Modified preS-Derived Peptides of HBV

As outlined above, the present invention provides hydrophobic modifiedpreS-derived peptides of hepatitis B virus (HBV).

The envelope of HBV encloses three proteins termed L (large), M (middle)and S (small) (see FIG. 1A). They share the C-terminal S-domain withfour transmembrane regions. The M- and L-protein carry additionalN-terminal extensions of 55 and, genotype-dependent, 107 or 118 aminoacids (preS2- and preS1) (see FIG. 1B).

Thus, the expression “preS-derived” peptide of HBV according to thepresent invention refers to a peptide with an amino acid sequence thatcorresponds to the N-terminal extensions of the L-protein of HBV, preS1,preferably to a consensus sequence of the species and genotypes A to Has well as of woolly monkey (WMHBV), chimpanzee and gorilla hepatitis Bviruses, but it also refers to variants thereof, preferably N-terminallyand/or C-terminally truncated variants, amino acid substitutionvariants.

SEQ ID NO. 1 shows the HBV preS consensus amino acid sequence of thespecies and genotypes A to H as well as of woolly monkey (WMHBV).

See the alignment in FIG. 2 showing the HBV preS consensus sequence(Consensus) and the eight HBV genotypes (A-H) as well as the woollymonkey HBV (WMHBV) preS sequence encompassing amino acids 2-48. Notethat the genotypes A, B, C, E, F, G and H have up to eleven additionalamino acids at their N-termini.

The amino acid sequence of HBV “genotype C” within this applicationrefers to an artificial sequence, which corresponds to or is identicalto the HBV Genotype C, as e.g. shown in Genbank ABV02850.1, except thatposition 46 (according to the numbering as described below) is Lys (K)in the genotype C of the present invention instead of Gln (Q) as in theGenbank sequence; the HBV genotype C sequence of this application canalso be referred to as “HBV genotype C Q46K”. See also SEQ ID NOs. 4,12, 21-27.

FIG. 2 also shows the numbering of the amino acid residues of the HBVpreS consensus sequence, which will be referred to throughout thisspecification:

Amino acid residue number 1 is the methionine (Met1) of genotype D(formerly described as subtype ayw, see also SEQ ID NO. 5), whereasamino acid residue number (−11) is the methionine (Met(−11)) of genotypeC (SEQ ID NO. 4). In vivo Met1 or Met(−11), respectively, is cleaved offby a cellular methionyl aminopeptidase and modified by a subsequenttransfer of a myristoyl residue from Myristoyl-CoA to amino acid residuenumber 2 glycine (Gly2) or amino acid residue number (−10) glycine(Gly(−10)), respectively, by N-myristoyl transferase. The N-terminalamino acid residue of genotype D is the natural amino acid Glycin (Gly2)and is numbered 2 according to the respective numbering from the codonsof the underlying open reading frame of L (or e.g. Gly(−10) for genotypeC).

The HBV preS consensus sequence also comprises the additional N-terminalamino acids of genotypes A, B, C, E, F, G and H (designated at “−”positions). Thus, in total the HBV preS consensus sequence encompassespositions (−11) to 48.

Thus, there is a difference between the above described numbering andthe actual listing of amino acids in SEQ ID NO. 1, e.g.

-   -   Met (−11), residue number (−11), is listed as amino acid residue        1 in SEQ ID NO. 1;    -   Gly (−10), residue number (−10), is listed as amino acid residue        2 in SEQ ID NO. 1;    -   Met 1, residue number 1, is listed as amino acid residue 12 in        SEQ ID NO. 1;    -   Gly 2, residue number 2, is listed as amino acid residue 13 in        SEQ ID NO. 1;    -   Gly 48, residue number 48, is listed as amino acid residue 58 in        SEQ ID NO. 1.

HBV preS consensus sequence (positions (−11) to 48) SEQ ID NO: 1(−11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRANSNNP DWDFN PNKDH WPEAN KVG-48 HBV Genotype A SEQ ID NO: 2(−11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFGANSNNP DWDFN PVKDD WPAAN QVG-48 HBV Genotype B SEQ ID NO: 3(−11)-M GGWSS KPRKG MGTNL SVPNP LGFFP DHQLD PAFKANSENP DWDLN PHKDN WPDAN KVG-48artificial amino acid sequence, which correspondsto HBV Genotype C except that position 46 is Lys(K) instead of Gln (Q); Q46K SEQ ID NO: 4(−11)-M GGWSS KPRQG MGTNL SVPNP LGFFP DHQLD PAFGANSNNP DWDFN PNKDH WPEAN KVG-48 HBV Genotype D SEQ ID NO: 51-MGQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVG-48HBV Genotype E SEQ ID NO: 6(−10)-MGLSW TVPLE WGKNI STTNP LGFFP DHQLD PAFRANTRNP DWDHN PNKDH WTEAN KVG-48 HBV Genotype F SEQ ID NO: 7(−11)-M GAPLS TTRRG MGQNL SVPNP LGFFP DHQLD PLFRANSSSP DWDFN TNKDS WPMAN KVG-48 HBV Genotype G SEQ ID NO: 8(−10)-MGLSW TVPLE WGKNL SASNP LGFLP DHQLD PAFRANTNNP DWDFN PKKDP WPEAN KVG-48 HBV Genotype H SEQ ID NO: 9(−11)-M GAPLS TARRG MGQNL SVPNP LGFFP DHQLD PLFRANSSSP DWDFN TNKDN WPMAN KVG-48 WMHBV SEQ ID NO: 101-MGLNQ STFNP LGFFP SHQLD PLFKA NAGSA DWDKN PNKDP WPQAH DTA-48

For SEQ ID NOs. 2-10 see also Genbank Accession numbers:

-   -   HBV Genotype A Genbank AAT28684.1    -   HBV Genotype B Genbank AAU01950.1    -   HBV Genotype C Genbank ABV02850.1 (wherein position 46 is        Lys (K) (as in SEQ ID NO. 4) instead of Gln (Q) (of ABV02850.1)    -   HBV Genotype D Genbank AAR19337.1    -   HBV Genotype E Genbank ABS31101.1    -   HBV Genotype F Genbank ABK19774.1    -   HBV Genotype G Genbank AAF34735.1/AF160501_3    -   HBV Genotype H Genbank AAM09052.1    -   WMHBV Genbank AAC16905.1

A hydrophobic modified preS-derived peptide of HBV according to thepresent invention has the formula[H_(m)-P-R_(n)]A_(o)

-   -   wherein    -   P is said preS-derived peptide;    -   H is said hydrophobic modification of P;    -   R is a C-terminal modification of P;    -   A is an anchor group;    -   m is at least 1;    -   n is 0 or at least 1;    -   o is 0 or at least 1.

Preferably, not both n and o are 0.

The preS-derived peptide of HBV, P, according to the present inventioncomprises:

-   -   the amino acid sequence of the HBV preS consensus sequence as        shown in SEQ ID NO. 1 or    -   variants thereof.

“Variants” are preferably N-terminally and/or C-terminally truncatedvariants, amino acid substitution or deletion variants, or prolongedvariants. Variants comprise furthermore an amino acid sequencecomprising modified amino acid(s), unnatural amino acid(s) orpeptidomimetic(s) or further compounds which can mimic a peptidebackbone/structure.

Preferably, variants are selected from N-terminally and/or C-terminallytruncated variants of SEQ ID NO. 1; amino acid substitution or deletionvariants; variants comprising modified amino acid(s), unnatural aminoacid(s) or peptidomimetic(s) or further compounds which can mimic apeptide backbone/structure.

According to the invention, a variant of a hydrophobic modifiedpreS-derived peptide contains at least 10 or 20 consecutive amino acidsof SEQ ID NO. 1 and can consist of 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more amino acids of SEQ IDNO. 1 or its variants.

N-terminally and/or C-terminally truncated variants comprise preferablyat least 14 consecutive amino acids (such as residues 5 to 18), morepreferably at least 19 consecutive amino acids (such as residues 2 to20), even more preferably at least 20 consecutive amino acids of SEQ IDNO. 1 (such as residues 2 to 21).

The term “variant” also refers to the homologous sequences found in thedifferent viral species, strains or subtypes of the hepadnavirus genus,such as HBV strain alpha1, HBV strain LSH (chimpanzee isolate), woollymonkey HBV (WMHBV), or strains selected from the group consisting of theHBV genotypes A to H, as well as the HBV genotype C as defined herein,Q46K (such as SEQ ID NO. 4).

The term “variant” also refers to homologous sequences which show atleast 50% sequence identity to SEQ ID NO. 1 or any other amino acidsequence disclosed herein, preferably 70%, more preferably 80%, evenmore preferably 90% or 95%.

Thus, in preferred hydrophobic modified preS-derived peptide accordingto the invention P comprises a variant of SEQ ID NO. 1 with an aminoacid sequence of the different viral species, strains or subtypes,preferably of the genotypes of HBV or woolly monkey HBV (WMHBV) orvariants thereof.

Preferably, P comprises an amino acid sequence selected from SEQ ID NOs.2 to 10 or variants thereof (see also FIG. 2).

In a preferred embodiment in the hydrophobic modified preS-derivedpeptides according to the invention P comprises a variant of SEQ ID NO.1 with an amino acid sequence selected from

-   -   amino acid residues 2 to 48 [SEQ ID NO. 11]    -   amino acid residues 2 to 20    -   amino acid residues 2 to 21 [SEQ ID NO. 12]    -   amino acid residues 9 to 15 [SEQ ID NO. 21]        of the HBV preS consensus sequence as shown in SEQ ID NO. 1.

More preferably, P does not comprise amino acid substitutions and/ordeletions at residues 9 to 22 of SEQ ID NO. 1, such as by deletingresidues 17 to 21.

More preferably, P does not comprise amino acid substitutions and/ordeletions at residues 9 to 15 of SEQ ID NO. 1.

More preferably, the sequence motif NPLGFFP (corresponding to residues 9to 15 of SEQ ID NO. 1) is not interrupted or modified, such as byreplacing residues 11-15 by D-amino acids.

The inventors have identified these amino acid residues to be importantfor the liver tropism of the hydrophobic modified preS-derived peptidesof HBV, as described below.

More preferably, in the hydrophobic modified preS-derived peptidesaccording to the invention P comprises a variant of SEQ ID NO. 1 with anamino acid sequence selected from

-   -   amino acid residues 2 to 48 of the consensus sequence [SEQ ID        NO. 11];    -   amino acid residues 2 to 21 of the consensus sequence [SEQ ID        NO. 12];    -   amino acid residues 2 to 48 of genotype C [SEQ ID NO. 13];    -   amino acid residues 5 to 48 of genotype C [SEQ ID NO. 14];    -   amino acid residues 9 to 48 of genotype C [SEQ ID NO. 15];    -   amino acid residues 2 to 21 of genotype C [SEQ ID NO. 16];    -   amino acid residues 5 to 21 of genotype C [SEQ ID NO. 17];    -   amino acid residues 9 to 21 of genotype C [SEQ ID NO. 18];    -   amino acid residues 2 to 15 of genotype C [SEQ ID NO. 19];    -   amino acid residues 5 to 15 of genotype C [SEQ ID NO. 20];    -   amino acid residues 9 to 15 of genotype C [SEQ ID NO. 21];    -   amino acid residues 2 to 48 of genotype D [SEQ ID NO. 23];    -   amino acid residues 5 to 48 of genotype D [SEQ ID NO. 24];    -   amino acid residues 9 to 48 of genotype D [SEQ ID NO. 25];    -   amino acid residues 2 to 21 of genotype D [SEQ ID NO. 26];    -   amino acid residues 5 to 21 of genotype D [SEQ ID NO. 27];    -   amino acid residues 9 to 21 of genotype D [SEQ ID NO. 28];    -   amino acid residues 2 to 20 of genotype D [SEQ ID NO. 38];    -   amino acid residues 2 to 15 of genotype D [SEQ ID NO. 29];    -   amino acid residues 5 to 15 of genotype D [SEQ ID NO. 30];    -   amino acid residues 9 to 15 of genotype D [SEQ ID NO. 21].

Thus preferably, P comprises an amino acid sequence selected from SEQ IDNOs. 11 to 30 and 38 or variants thereof.

“Variants” of SEQ ID NO. 1 also comprise variants or “analogues”comprising amino acid deletions, amino acid substitutions, such asconservative or non conservative replacement by other amino acids or byisosteres (modified amino acids that bear close structural and spatialsimilarity to protein amino acids), amino acid additions or isostereadditions, as long as the sequence still shows liver tropism, preferablymore than 10% of the injected dose/g liver tissue after 1 h.

Conservative amino acid substitutions typically relate to substitutionsamong amino acids of the same class. These classes include, for example,

-   -   amino acids having uncharged polar side chains, such as        asparagine, glutamine, serine, threonine and tyrosine;    -   amino acids having basic side chains, such as lysine, arginine,        and histidine;    -   amino acids having acidic side chains, such as aspartic acid and        glutamic acid; and    -   amino acids having nonpolar side chains, such as glycine,        alanine, valine, leucine, isoleucine, proline, phenylalanine,        methionine, tryptophan, and cysteine.

Preferably, the hydrophobic modified preS-derived peptides of theinvention are modified to decrease their immunogenic properties.

Thus, preferred sequence motifs to be modified by substitution and/ordeletion are sequences responsible for the immunogenicity, such asB-cell epitopes and/or T-cell epitopes.

B-cell epitopes of HBV are preferably amino acid residues 20 to 23,motif DPAF, of SEQ ID NO. 1 and amino acid residues 26 to 32, motifNSNNPDW of the consensus sequence (SEQ ID NO. 1) and genotype C (SEQ IDNO. 4) or NTANPDW of genotype D (SEQ ID NO. 5).

In a preferred embodiment amino acid residues 20 to 23 of SEQ ID NO. 1are modified, preferably by amino acid substitution, and/or the aminoacid residues 26 to 32 are modified, preferably by amino acidsubstitution.

Preferably, amino acid residues 20 to 23 of SEQ ID NO. 1 (motif DPAF)are modified by alanine amino acid substitution, preferably into motifAPAF.

Preferably, amino acid residues 26 to 32 of SEQ ID NO. 1 are modified byalanine amino acid substitution, preferably into motif NANAPDW orNAAAPDW.

Further preferred sequence motifs to be modified by substitution and/ordeletion are sequences that are antibody recognition motifs.

A preferred objective for modifying sequences responsible for theimmunogenicity, such as B-cell epitopes, is decreasing theimmunogenicity of the hydrophobic modified preS-derived peptides of theinvention. The person of skill in the art is aware of this objective andits advantages and disadvantages. Importantly, while decreasing theimmunogenicity of the hydrophobic modified preS-derived peptides of theinvention one has also to consider the desired liver targeting/tropismfeature. Shuttle peptides that do not elicit immune responses can berepeatedly administered without systemic removal from humoral immuneresponses.

Thus, in a preferred embodiment P does not comprise amino acidsubstitutions and/or deletions at residues 9 to 15 of SEQ ID NO. 1 inorder to maintain the liver targeting/tropism.

A more preferred hydrophobic modified preS-derived peptide of thisinvention shows a high liver targeting/tropism and low or noimmunogenicity.

In a preferred embodiment P comprises an amino acid sequence selectedfrom SEQ ID NOs. 31 to 37 or variants thereof.

See also Tables and the respective Figures and Examples.

In a more preferred embodiment P comprises an amino acid sequenceselected from

-   -   SEQ ID NO. 11 (residues 2 to 48 of the consensus sequence),    -   SEQ ID NO. 12 (residues 2 to 21 of the consensus sequence),    -   SEQ ID NO. 13 (residues 2 to 48 of genotype C),    -   SEQ ID NO. 16 (residues 2 to 21 of genotype C),    -   SEQ ID NO. 23 (residues 2 to 48 of genotype D),    -   SEQ ID NO. 26 (residues 2 to 21 of genotype D),    -   SEQ ID NO. 38 (residues 2 to 20 of genotype D), and    -   SEQ ID NO. 31 (residues 2 to 48 of genotype C and Ala mutations        in positions 21, 23, 29 and 30).

The above are the preferred amino acid sequences which fulfill thepreferred combination of the desired features: short amino acidsequence, low or no immunogenicity and efficient liver targeting.

The hydrophobic modification (H) of the preS-derived peptide P isN-terminal of P.

“N-terminal” refers to the hydrophobic modification at the N-terminus,i.e. the respective first amino acid residue (e.g. Gly 2), but comprisesalso the hydrophobic modification in close proximity to the N-terminus,such as respective amino acid residues (−4), (−3), (−2), (−1), 1, 2 or 3or 4. Thus, the hydrophobic modification can furthermore be obtained byan attachment of a hydrophobic moiety at a site close to the N-terminusof P.

The hydrophobic modification of said preS-derived peptide of HBVaccording to the present invention adds a hydrophobic moiety to thepeptide.

Furthermore, m is at least 1, i.e. modification with at least onehydrophobic moiety or group.

In preferred embodiments of this invention m is 1, 2, 3, 4 or more. Thatis, P can be modified with more than one hydrophobic moiety or group,such as 2. The hydrophobic moieties or groups can be the same ordifferent to each other.

The hydrophobic modification of said preS-derived peptide of HBVaccording to the present invention is selected from:

-   -   acylation;    -   addition of hydrophobic moieties.

Acylation is preferably selected from acylation with carboxylic acids,fatty acids, amino acids with lipophilic side chains.

Preferred fatty acids are saturated or unsaturated fatty acids, branchedor unbranched fatty acids, preferably with 8 to 22 carbon atoms (C8 toC22).

More preferably, the hydrophobic modification by acylation is selectedfrom acylation with myristoyl (C14), palmitoyl (C16) or stearoyl (C18).

Modification by myristoylation is preferred in in vivo and medicinalapplications due to its higher safety, e.g. not showing the adverseeffects of the stearoyl group (innate immune response etc).

The addition of hydrophobic moieties is preferably selected fromaddition of cholesterol, derivatives of cholesterol, phospholipids,glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives,adamantane, farnesol, aliphatic groups, polyaromatic compounds.

The attachment of the hydrophobic moieties is preferably by covalentbinding, which can be achieved via carbamate, amide, ether, disulfide orany other linkage that is within the skill of the person skilled in theart.

Thus, the hydrophobic modified, preferably acylated preS-derivedpeptides of this invention are preferably lipopeptides due to theirN-terminal lipophilic or hydrophobic group/moiety.

The C-terminal modification (R) of said preS-derived peptide P ispreferably a modification with a moiety that protects from degradation,such as in vivo degradation.

“C-terminal” refers to the modification at the C-terminus, i.e. therespective last amino acid residue, but comprises also the modificationin close proximity to the C-terminus, such as the last but one aminoacid residue, the last but two amino acid residue or more amino acidresidues (e.g. introduction of one D-amino acid that protects thecarrier from enzymatic degradation e.g. by the action ofcarboxypeptidases).

The skilled artisan will be able to select the respective suitablemoiety(s) depending on the respective application.

Preferred moieties that protect from degradation are selected fromamides, D-amino acids, modified amino acids, cyclic amino acids,albumin, natural and synthetic polymers, such as PEG, glycane.

Furthermore, n is 0 or at least 1, i.e. the C-terminal modification (R)is optional.

Preferably, n is 1.

In further embodiments of this invention n is 1, 2, 3, 4 or more. Thatis, the C-terminus of P or its proximity can be modified with more thanone moiety or group, such as 2. The moieties or groups can be the sameor different to each other.

In an embodiment of this invention H and/or R are linked to P via alinker or spacer.

Linker or spacer are known to the skilled artisan, such as polyalanine,polyglycin, carbohydrates, (CH₂)_(n) groups.

The skilled artisan will, thus, be able to select the respectivesuitable linker(s) or spacer(s) depending on the respective application.

The anchor group (A) serves as a point of attachment for a compound, atag, a label.

In a preferred embodiment, the anchor group is “C-terminal” of P,wherein “C-terminal” refers to the modification at the C-terminus, i.e.the respective last amino acid residue, but comprises also the closeproximity of the C-terminus, such as the last but one amino acidresidue, the last but two amino acid residue or more amino acidresidues. In this case n can be 0, thus there is no other C-terminalmodification R.

In this embodiment, the hydrophobic modified preS-derived peptide of HBVcan have the following general formula

The anchor group A can be at an amino acid side chain of thepreS-derived peptide P or can be the amino acid side chain of thepreS-derived peptide P itself, i.e. A can be a side chain itself or amodified side chain.

The anchor group can also be a modified amino acid residue which wasintroduced into the amino acid sequence of P to serve as an anchorgroup.

In other embodiments of the invention the anchor group A is attached tothe hydrophobic modification H and/or the C-terminal modification R.

Preferred anchor groups are selected from ester, ether, disulfide,amide, thiol, thioester.

The skilled artisan will be able to select the respective suitableanchor group(s) depending on the respective compound, tag, label etc. tobe attached.

The anchor group can furthermore be suitable for attaching acomplex-forming component, such as of the biotin/avidin,polyarginine/oligonucleotide (e.g. siRNA) complex.

Furthermore, o is 0 or at least 1, i.e. the anchor group (R) isoptional.

Preferably, o is 1.

In further embodiments of this invention o is 1, 2, 3, 4 or more. Thatis, there are more than one anchor group, such as 2. The anchor groupscan be the same or different to each other, allowing the attachment ofseveral compounds, such as a drug and a label or different drugs.

For preferred hydrophobic modified preS-derived peptides, see alsoTables 1 and 2.

More preferred hydrophobic modified preS-derived peptides of theinvention are the following:

-   -   P comprises an amino acid sequence selected from SEQ ID NO. 11,        SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 16, SEQ ID NO. 23, SEQ        ID NO. 26, SEQ ID NO. 31 and SEQ ID NO. 38, and    -   H is a hydrophobic modification by acylation with stearoyl (C18)        or myristoyl (C14).

Thus, the more preferred hydrophobic modified preS-derived peptides ofthe invention are:

Amino acid Hydrophobic Designation of peptide sequence modificationHBVpreS/2-48^(stearoyl) (consensus) SEQ ID NO. 11 Stearoyl (C18)HBVpreS/2-48^(stearoyl) (consensus) SEQ ID NO. 12 Stearoyl (C18)HBVpreS/2-48^(stearoyl) (C) SEQ ID NO. 13 Stearoyl (C18)HBVpreS/2-48^(stearoyl) (C) SEQ ID NO. 16 Stearoyl (C18)HBVpreS/2-48^(myr) (C) SEQ ID NO. 13 Myristoyl (C14) HBVpreS/2-48^(myr)(D) SEQ ID NO. 23 Myristoyl (C14) HBVpreS/2-21^(stearoyl) (D) SEQ ID NO.26 Stearoyl (C18) HBVpreS/2-20^(myr) (D) SEQ ID NO. 38 Myristoyl (C14)HBVpreS/2-48^(stearoyl)- SEQ ID NO. 31 Stearoyl (C18) Ala^(21,23,29,30)(C) Wherein (C) refers to HBV genotype C Q46K.

The peptides of this invention can be prepared by a variety ofprocedures readily known to those skilled in the art, in general bysynthetic chemical procedures and/or genetic engineering procedures.

Synthetic chemical procedures include more particularly the solid phasesequential and block synthesis (Erickson and Merrifield, 1976). Thesolid phase sequential procedure can be performed using establishedautomated methods such as by use of an automated peptide synthesizer. Inthis procedure an α-amino protected amino acid is bound to a resinsupport. The resin support employed can be any suitable resinconventionally employed in the art for the solid phase preparation of(poly)peptides, preferably polystyrene which has been copolymerized withpolyoxyethylen to provide sites for ester formation with the initiallyintroduced o-amino protected amino acid. This optimized method, appliedby the inventors, has been explicitly described (see e.g. 12). The aminoacids are introduced one by one (step-wise). Each synthesis cyclecorresponding to the introduction of one amino acid includes adeprotection step, successive washing steps, a coupling step withactivation of the amino acid, and subsequent washing steps. Each ofthese steps is followed by a filtration. The reactive agents forcoupling are the classical reactive agents for (poly)peptide synthesissuch as dicyclohexylcarbodiimide, hydroxybenzotriazole,benzotriazil-1-yl-oxytris (dimethylamino) phosphoniumhexafluorophosphate, and diphenylphosphorylazide. After synthesis of thepolypeptide on the resin, the polypeptide is separated from the resin bya treatment with a strong acid such as trifluoroacetic acid in thepresence of anisol, ethanedithiol or 2-methylindole. The compound isthen purified by the classical techniques of purification, in particularby means of HPLC.

The peptides of the present invention may also be obtained by coupling(poly)peptide fragments that are selectively protected, this couplingbeing effected e.g. in a solution.

The peptides can further be produced by genetic engineering techniquesas known to the skilled artisan. An eukaryotic expression system, suchas the baculovirus system, is particularly suitable. According to thisprocedure proteins are expressed in insect cells infected with arecombinant baculovirus containing a nucleic acid sequence encoding aheterologous protein and regulating nucleic acid sequences, such as apromoter. Several cell-lines are available for infection withrecombinant baculovirus, such as cell line Sf-9, available from theAmerican Type Culture Collection (CRL 1711). Expression in prokaryoticexpression system, such as E. coli, is also particularly suitable.

The introduction of the hydrophobic moiety to the peptide can beaccomplished by a variety of procedures readily known to those skilledin the art, including synthetic and genetic engineering approaches.

Alternatively, the peptides and/or fusion peptides (i.e. hydrophobicmodified peptides) can be produced by stably transfected eukaryotic celllines, like CHO and other cell lines which are known in the art andusually used for generating vaccines and the like. Due to the intrinsicproperty that the N-terminal 47-preS1 amino acids promote secretion of amyristoylated protein/peptide, the biologically active hydrophobicmodified peptide can be extracted from cell culture supernatants.

Vectors and Shuttles for Liver Targeting

As outlined above, the present invention provides the use of thehydrophobic modified preS-derived peptides of HBV as vehicle or shuttlefor the specific delivery of a compound to the liver.

“Vehicle” or “shuttle” for the specific delivery of a compound to theliver according to the present invention refers to the liver tropism orhepatotropism of the hydrophobic modified preS-derived peptides of HBVas found by the inventors and described herein, i.e. to their capacityto selectively accumulate in the liver, preferably to selectivelyaccumulate at the plasma membrane of hepatocytes as well as toselectively enter into hepatocytes.

The invention is based on the finding of a highly specific liveraccumulation and on the identification of the determinants of the livertropism of HBV in the preS1 sequence of HBV by the inventors. Thus, theinvention uses the knowledge about the determinants of the liver tropismfor the design of universal vehicles or shuttles for specific livertargeting or delivery, respectively.

The hydrophobic modified preS-derived peptides of the present inventionare versatile vehicles or shuttles for specifically deliveringcompound(s) to the liver.

Preferably, the specific delivery of a compound to the liver is thespecific delivery of the compound to hepatocytes.

Furthermore, the compound can specifically be delivered to hepatocytesin vitro as well as in vivo.

The compound is preferably specifically delivered to the liver of ananimal, preferably mammal or human, or a bird.

Compounds to be Delivered

The “compound” to be specifically delivered to the liver according tothis invention can be any kind of compound.

Preferred compounds are drugs and/or labels.

Drugs can be in form of prodrugs or preprodrugs.

A compound can also be a virus or derivatives thereof, such as areplication-deficient or a replication-competent recombinant virus (e.g.an Adenovirus or an Adeno-associated virus) that has been chemically orgenetically modified to expose the targeting sequence on its surface andis thereby redirected to infect hepatocytes. These viruses shall beapplicable for hepatocyte specific gene delivery.

In a preferred embodiment of the invention the compound to bespecifically delivered to hepatocytes is a drug (or a drug in form of aprodrug) and is preferably selected from

Drug class Examples interferon IFNα viral reverse transcriptaseinhibitor Lamivudine, Adefovir, Entecavir viral RNA polymerase inhibitorHCV NS5B polymerase inhibitor viral core assembly inhibitor or forhepatropic viruses viral nucleocapsid inhibitor e.g.dihydroarylpyrimidine-derived HBV capsid assembly inhibitors kinaseinhibitor Raf kinase inhibitor Sorafenib (BAY 43-9006) nucleosideanalogue protease inhibitor - HCV NS3 protease inhibitor, BILN- viral orgeneral protease inhibitor 2061, VX950 proteasom inhibitor MG132,bortezomib antibody or fragment thereof Neutralizing anti-HCV E2antibodies siRNA or precursor thereof HBV, HCV, HDV-specific siRNAs andmodifications thereof; siRNAs targeting other sequences, includingcellular sequences farnesylation inhibitor; such as farnesyl transferaseinhibitor (FTI) alcohol dehydrogenase (ADH) or activator thereofcholesterol biosynthesis inhibitor, Mevinacor/Lovastatin such asHMD-CoA-reductase inhibitors inhibitor of the liver stage of aPrimaquine virus or a non-viral pathogen, such as malaria,schistosomiasis (bilharziosis), leishmaniasis inhibitors of otherviruses that e.g. Zovirax for the treatment of occasionally infect theliver herpes virus infection antibiotics to specifically act in theliver

The compound is preferably selected from a clinically approved drug.

In an embodiment the compound to be delivered is IFN-alpha.

The hydrophobic modified preS1-derived peptide(s) used in such anembodiment can be HBVpreS/2-20^(myr)(D), HBVpreS/2-48^(myr)(D),HBVpreS/2-20^(stearoyl)(D), HBVpreS/2-48^(stearoyl)(D) (see also FIG.14). A fusion protein or construct with mouse interferon-alpha wasobtained by recombinant expression in insect cells using the baculovirusexpression system.

See also FIGS. 14 and 15 as well as Examples.

In a preferred embodiment of the invention the compound to bespecifically delivered to hepatocytes is a label and is preferablyselected from a fluorescent dye, a radioisotope and a contrast agent.

Preferred radioisotopes are ¹³¹I, ¹²⁵I, ^(99m)Tc, ¹⁸F, ⁶⁸Ga, ¹¹¹In, ⁹⁰Y,¹⁷⁷Lu.

Preferred fluorescent dyes are Alexa dyes, derivatives of rhodamine andfluorescein, Cy-dyes, FITC.

Preferred contrast agents are Gadolinium (Gd) complexes, supramagneticiron (Fe) complexes and particles, compounds containing atoms of highatomic number, i.e. iodine for computer tomography (CT), microbubblesand carriers such as liposomes that contain these contrast agents.

In a further preferred embodiment of the present invention also thecompound is labelled, i.e. carries a label as defined herein.

In preferred embodiments the compounds are in form of depots orcarriers, which e.g. carry a drug, prodrug or label. Such depots orcarriers are known in the art, such as nanoparticles, liposomes,microbubbles, gas emulsions.

In other embodiments of the present invention the compound to bedelivered to the liver is selected from immunogenic epitopes. Preferredimmunogenic epitopes when delivered to the liver servehepatocyte-mediated antigen presentation in order to activateliver-directed immunological responses.

In a preferred embodiment the compound and the hydrophobic modifiedpreS-derived peptide of HBV form a conjugate.

Preferably, the conjugate of compound and hydrophobic modifiedpreS-derived peptide is formed by covalent attachment or by complexformation.

Preferably, in a conjugate the compound is covalently attached to thehydrophobic modified preS-derived peptide, preferably by attaching thecompound to an anchor group A. The form of attachment depends on thetype of compound. The person of skill in the art will be able todetermine suitable anchor groups for forming suitable conjugates.

Preferably, A furthermore comprises a spacer or linker.

The spacer or linker preferably comprises a recognition site forhepatocyte specific activation, which is preferably recognized by aliver protein.

The recognition site is preferably a proteolytic cleavage site. Theliver protein is, thus, preferably a hepatocellular protein, morepreferably a hepatocellular proteolytic enzyme. Thus, the conjugate canbe administered to a subject and will be transported through the body,such as in the bodily fluids, without being cleaved. However, as soon asthe conjugate reaches its target, the liver or the hepatocytes,respectively, the liver protein, such as a hepatocellular proteolyticenzyme will cleave the proteolytic cleavage site and release thecompound from its shuttle, i.e. the hydrophobic modified preS-derivedpeptide.

Further preferred liver proteins are cytochromes, such as cytochromeP450. The HepDirect® technology (of Metabasis Technologies, Inc.) asused in Adefovir or Pradevofir, is also suitable for the presentinvention.

In one embodiment the conjugate of compound and hydrophobic modifiedpreS-derived peptide is formed by complex formation. Preferred complexesuseful in the invention are biotin/avidin, polyarginine/oligonucleotide(e.g. siRNA). The skilled artisan will be able to determine suitablecomplex components and to design the compound and hydrophobic modifiedpreS-derived peptide accordingly.

The hydrophobic modified preS-derived peptide, in particular theconjugates of the present invention, are preferably used to enrich acompound, that is shuttled to the liver, in the liver.

Preferably, the compound is cleaved of the conjugate with thehydrophobic modified preS-derived peptide of HBV by a liver protein,preferably a hepatocellular proteolytic enzyme, in particular in vivo inthe liver.

Diagnosis, Prevention and/or Treatment of Liver Diseases

In a preferred embodiment of the invention the above hydrophobicmodified preS-derived peptides, in particular their conjugates withcompounds, are provided for the diagnosis, prevention and/or treatmentof a liver disease or disorder.

Depending on the liver disease or disorder which shall be diagnosed,prevented and/or treated the respective compound is selected andselectively, specifically delivered to the liver.

A “liver disease” or a “liver disorder” according to the presentinvention refers to any disease or disorder that has an effect on orinvolves the organ liver, liver tissue or hepatocytes.

Examples of liver diseases are:

-   -   Hepatitis: inflammation of the liver, caused mainly by various        viruses but also by certain poisons, autoimmunity or hereditary        conditions;    -   Cirrhosis: the formation of fibrous tissue in the liver,        replacing dead liver cells. The death of the liver cells can for        example be caused by viral hepatitis, alcoholism or contact with        other liver-toxic chemicals;    -   Haemochromatosis: a hereditary disease causing the accumulation        of iron in the body, eventually leading to liver damage;    -   Cancer of the liver: primary hepatocellular carcinoma (HCC) or        cholangiocarcinoma and metastatic cancers, usually from other        parts of the gastrointestinal tract;    -   Wilson's disease: a hereditary disease which causes the body to        retain copper;    -   Primary sclerosing cholangitis: an inflammatory disease of the        bile duct, autoimmune in nature;    -   Primary biliary cirrhosis: autoimmune disease of small bile        ducts;    -   Budd-Chiari syndrome: obstruction of the hepatic vein;    -   Gilbert's syndrome: a genetic disorder of bilirubin metabolism,        found in about 5% of the population;    -   Glycogen storage disease type II: the build-up of glycogen        causes progressive muscle weakness (myopathy) throughout the        body and affects various body tissues, particularly in the        heart, skeletal muscles, liver and nervous system;    -   pediatric liver disease, such as biliary atresia,        alpha-1-antitrypsin deficiency, alagille syndrome, and        progressive familial intrahepatic cholestasis;    -   metabolic diseases.

Furthermore, also liver diseases of animals, such as pets or livestock,are included, in particular diseases that can be transmitted to humans,such as toxoplasmosis.

The liver disease or disorder to be diagnosed, prevented and/or treatedis preferably selected from hepatitis, cirrhosis, haemochromatosis,preferably hepatitis caused by hepatitis A, B, C, D, E, F, G and Hvirus.

The liver disease or disorder to be diagnosed, prevented and/or treatedcan also be concomitant hepatitis caused by viruses, such as viruses ofthe family Herpesviridae, e.g. herpes virus, cytomegalie virus (CMV) butalso varicella zoster virus (VZV), Epstein Barr virus (EBV), coxsackieviruses, yellow fever virus, Dengue virus.

The liver disease or disorder to be diagnosed, prevented and/or treatedcan also be a disease which involves a liver stadium of a virus or anon-viral pathogen, such as in many tropical diseases. Since the liverstadium of some pathogens is an early stadium, the respective infectioncan be selectively, specifically treated in such an early stadium.

Such viruses are hepatitis A, B, C, D, E, F, G and H virus, herpesviruses.

Such non-viral pathogens are bacteria, parasites and/or worms.

Parasites are for example protozoan parasites of the genus Plasmodiumthat cause malaria, such as Plasmodium falciparum, Plasmodium vivax, andrelated species (e.g. Plasmodium ovale, Plasmodium malariae, Plasmodiumknowlesi).

Such worms are for example flatworms of the genus Schistosoma that causeschistosomiasis or bilharziosis, such as Schistosoma mansoni,Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicumand Schistosoma mekongi.

Such parasites are also for example Leishmania trypanosome protozoa ofthe genus Phlebotomus and Lutzomyia which are responsible for thedisease leishmaniasis.

Therefore, malaria, schistosomiasis (bilharziosis), and/or leishmaniasiscan be diagnosed, prevented and/or treated by the means of thisinvention.

Therefore, certain tropical diseases can be diagnosed, prevented and/ortreated by the means of this invention.

The liver disease or disorder to be diagnosed, prevented and/or treatedare preferably liver tumors, preferably hepatocellular carcinoma (HCC).

The liver disease or disorder to be diagnosed, prevented and/or treatedcan also be a metabolic disease, such as diabetes, hyperlipidemia,metabolic syndrome and obesity, chronic hyperglycemia, metabolicsyndrome, non-alcoholic steatohepatitis (NASH) (see also (9)).

In a preferred embodiment of the invention the above hydrophobicmodified preS-derived peptides, in particular their conjugates withcompounds, are provided for the regulation of liver function.

Preferred is their use for hepatocyte-mediated antigen presentation andactivation of liver-directed immunological responses. In this case, thecompound to be delivered to the liver is preferably an immunogenicepitope.

In a preferred embodiment of the invention the above describedhydrophobic modified preS-derived peptides, in particular theirconjugates with compounds, are also suitable for the prevention ofhepatitis B and/or D infection. This is possible due to the property ofthe above described hydrophobic modified preS-derived peptides to targetnot only the liver (liver tropism) but furthermore act as viral entryinhibitors of HBV and/or HDV, which can be seen e.g. in FIG. 9. Thus,the above described hydrophobic modified preS-derived peptides aresuitable HBV and/or HDV vaccines. (See also corresponding PCT patentapplication of the inventors with the title: “Hydrophobic modifiedpreS-derived peptides of hepatitis B virus (HBV) and their use as HBVand HDV entry inhibitors”, which was filed at the same day.)

Thus, the above described acylated preS-derived peptides, in particulartheir conjugates with compounds, can be provided for the combineddiagnosis, prevention and/or treatment of a liver disease or disorderand the prevention of hepatitis B and/or D infection.

In a preferred embodiment of the invention the above described acylatedpreS-derived peptides, in particular their conjugates with compounds,can be used for the manufacture of a medicament for the diagnosis,prevention and/or treatment of a liver disease or disorder.

Pharmaceutical Compositions

As outlined above, the present invention provides a pharmaceuticalcomposition comprising at least one hydrophobic modified preS-derivedpeptide of HBV as defined herein and at least a compound to bespecifically delivered to the liver as defined herein and optionally apharmaceutically acceptable carrier and/or excipient.

The pharmaceutical composition according to the present inventioncomprises:

-   -   at least one hydrophobic modified preS-derived peptide of HBV as        defined herein above;    -   at least a compound to be specifically delivered to the liver,        preferably to hepatocytes, as defined herein above;        or    -   a conjugate as defined herein;        and    -   optionally a pharmaceutically acceptable carrier and/or        excipient.

The pharmaceutical compositions according to the present invention arevery well suited for all the uses and methods described herein.

A “pharmaceutically acceptable carrier or excipient” refers to anyvehicle wherein or with which the pharmaceutical or vaccine compositionsaccording to the invention may be formulated. It includes a salinesolution such as phosphate buffer saline. In general, a diluent orcarrier is selected on the basis of the mode and route ofadministration, and standard pharmaceutical practice.

Treatment Method

Furthermore, and as outlined above, the present invention providesmethods for the diagnosis, prevention and/or treatment of a liverdisease or disorder by utilizing the hydrophobic modified preS-derivedpeptide(s) of HBV or the pharmaceutical composition(s) of the invention.

The present invention also provides a method for the combined diagnosis,prevention and/or treatment of a liver disease or disorder and theprevention of HBV and/or HDV infection by administering to a subject aconjugate as defined herein, which comprises a hydrophobic modifiedpreS-derived peptide of HBV and a compound, or a pharmaceuticalcomposition as defined herein.

The method for the combined diagnosis, prevention and/or treatment of aliver disease or disorder and the prevention of hepatitis B and/or Dinfection according to the present invention comprises

-   -   administering to a subject in a therapeutically effective amount    -   (a) a conjugate comprising an hydrophobic modified preS-derived        peptide as defined herein above and at least a compound as        defined herein above, wherein the conjugate is as defined herein        above; or    -   (b) a pharmaceutical composition as defined herein above.        Route of Administration

Preferably, the route of administration of the conjugates orpharmaceutical compositions of the present invention, in particular inthe method of treatment, is selected from subcutaneous, intravenous,oral, nasal, intramuscular, transdermal, inhalative, by suppository.

A preferred embodiment for nasal administration or application is anasal spray.

Therapeutically Effective Amount

A “therapeutically effective amount” of a conjugate or a pharmaceuticalcomposition of this invention refers to the amount that is sufficient todiagnose, prevent and/or treat the respective liver disease or disorderand/or to prevent hepatitis B and/or D infection.

A preferred therapeutically effective amount is in the range of 10 μg to1 mg per kg body weight.

For the use of an hydrophobic modified preS-derived peptide of theinvention as a vaccine the preferred therapeutically effective amount isin the range of 10 μg to 1 mg per kg body weight.

In case of an IC₅₀ value of the hydrophobic modified preS-derivedpeptide used of about 10 nM, a preferred therapeutically effectiveamount is about 100 μg per kg body weight or in the range of 1 to 5 mgper patient.

The preferred therapeutically effective amount depends on the respectivecompound that is to be delivered and its respective therapeuticallyeffective amount.

The skilled artisan will be able to determine suitable therapeuticallyeffective amounts.

Gene Therapy Application

The present invention also provides the preS-derived peptides, i.e.nucleic acids encoding them, in gene therapy approaches.

A viral vector is provided that comprises a nucleic acid encoding apreS-derived peptide P as defined herein for the gene therapy of a liverdisease or disorder. The nucleic acid encodes the amino acid sequence ofthe preS-derived peptide P, which is necessary and sufficient toaccomplish liver targeting.

Preferably, the viral vector is replication defective, preferably anadeno-associated viral vector.

In an embodiment the viral vector further comprises a heterologoussequence to be expressed in the liver.

The viral vector is preferably a gene therapeutically suitable vector,which are known to the skilled person.

Identification of the HBV Receptor

The invention further relates to a method for in vitro and/or in vivoidentification of a hepatocyte receptor involved in the attachmentand/or penetration of HBV and/or quantitation of the expression of saidreceptor that comprises using a hydrophobic modified preS-derivedpeptide as described above.

Said hepatocyte receptor can be identified in mammals or respectiveanimal models, preferably mouse or human.

In particular, said method comprises the steps comprising:

-   -   contacting a liver biopsy or a hepatocyte with a hydrophobic        modified preS-derived peptide of the invention under conditions        and for a period of time sufficient to allow specific binding of        said peptide to a receptor expressed at the surface of a        hepatocyte;    -   detecting binding of said peptide to a receptor; and    -   identifying said receptor.

The method for the identification of a HBV receptor comprises the use of

-   -   (a) a conjugate comprising a hydrophobic modified preS-derived        peptide as defined herein and at least a compound as defined        herein, wherein the conjugate is as defined herein; or    -   (b) a pharmaceutical composition as defined herein.

This can be achieved according to classical procedures well-known by theskilled in the art. For instance, this could involve radioactive, enzymeor fluorescent labelling of the hydrophobic modified preS-derivedpeptides of the invention, and subsequent detection with an appropriatemethod. A number of fluorescent materials are known and can be utilizedas labels. These include, for example, fluorescein, rhodamine, auramine,Texas Red. Enzyme labels comprise conjugation of an enzyme to a moleculeof interest, e.g. a polypeptide, and can be detected by any ofcolorimetric, spectrophotometric, or fluorospectrophotometrictechniques.

The human Hepatitis B Virus (HBV) is the most important etiologicalfactor of hepatocellular carcinoma. Hallmarks of HBV infection are aremarkable in vivo efficacy and a pronounced liver tropism. The latteris probably the result of a specific interaction of one of the three HBVsurface proteins with liver associated receptors or liver-targetingfactors in the circulation system.

Based on recent insights into molecular details of the HBV entry pathwayand the identification of HBV-surface protein domains which areresponsible for hepatocyte binding and liver tropism, the presentinvention led to the development of a new class of peptide-drugconjugates to target liver diseases, such as HCC, and optionally tointerfere with HBV and HDV infection.

The inventors identified HBV-preS1-surface protein-derived lipopeptidesthat efficiently block HBV entry in vitro and in vivo. Biodistributionstudies of the present invention on these inhibitory peptides revealedthat they selectively accumulate in the liver were they bind to andpresumably enter into hepatocytes. This hepatotropism requiresN-terminal acylation of the peptide and depends on a certain HBVpreS-sequence motif within the N-terminal 47 preS1 amino acids, i.e.within the amino acid residues 2 to 21 or amino acid residues 2 to 20(or preferably the minimal sequence of residues 9 to 15). The inventors'observation that this peptide sequence additionally bears a membranetranslocation signal which facilitates the transport of even completefusion proteins across plasma membranes (unpublished results) opens thepossibility of specifically delivering any kind of drug to the plasmamembrane of hepatocytes or selectively even into this cell.

The inventors have shown that HBV preS1-derived lipopeptides are capableto completely prevent HBV infection in a transplanted uPA-RAG-1 mousemodel at very low doses. Pharmakokinetic studies on theseHBVpreS-derived entry inhibitors indicated a remarkable hepatotropismcombined with an extraordinary high serum stability (t_(1/2) ca. 60 h)and a long half life time in the target organ (t_(1/2) ca. 24 h). BothN-terminal acylation as well as the integrity of the certain amino acidsequence of the peptides are mandatory (i.e. within the amino acidresidues 2 to 21 (or preferably the minimal sequence of residues 9 to15)). The peptides can be used as versatile vectors for liver specificdrug targeting to conquer infections of hepatocytes or to treathepatocellular carcinoma.

The inventors have furthermore proven the principle that WMHBV infectioncan be efficiently blocked through subcutaneous application of HBVenvelope protein-derived peptides in vivo. This opens new perspectivesfor the prevention of acute HBV-infection and therapeutic options forchronic hepatitis B. Since the uPA/RAG-2/Pfp mice used lack B cells, Tcells, and NK cells, a direct inhibitory effect of the peptides onsusceptible hepatocytes is assumed. This is supported by the efficientaccumulation of acylated preS-derived peptides in the liver, followed bya slow clearance possibly via the biliary route. Both properties permitsubcutaneous application at very low doses and low frequencies. Giventhat 5 injections of 0.2 mg/kg HBV/preS2-48^(myr) within 5 days resultedin the prevention of the establishment of WMHBV infection, continuousadministration of the about 30-fold more active peptideHBV/preS2-48^(stearoyl) might be effective at doses below 7 μg/kg≈13nmol/kg when given daily or every 2 days. Taking into account that theefficient pharmacological dose per body weight obtained in mice has tobe corrected for humans (8) by a factor of about 10, the efficient doseper person is expected to be lower than 100 μg/day.

The inventors have also shown that

-   -   the HBV preS1-derived lipopeptides of the invention bind to        primary human hepatocytes (PHH) and that this binding is        myristoyl-dependent and sequence specific (see also FIG. 10),    -   Binding of the HBV preS1-derived lipopeptides of the invention        is not restricted to HBV-susceptible hepatocytes (see also FIG.        11),    -   Binding of the HBV preS1-derived lipopeptides of the invention        depends on the differentiation state of the cells and that they        only bind to differentiated hypocytes (see FIG. 12),    -   the HBV preS1-derived lipopeptides of the invention do not bind        to other hepatoma and non-hepatoma cell lines        -   no binding was seen to Hep G2 cells, Hep G2.215 cells, HuH 7            cells and CHO K1 cells (data not shown)

Thus, cellular factors addressed by the peptides depend on thedifferentiation state. But these are not critical for the restrictedhost-range of the virus.

TABLE 1 Preferred hydrophobic modified preS-derived peptides Designationof peptide Amino acid sequence HBVpreS/(−11)-48(consensus) consensus SEQID NO. 1 HBVpreS/2-48^(myr)(consensus) SEQ ID NO. 11HBVpreS/2-48^(stearoyl)(consensus) HBVpreS/2-21^(myr)(consensus) SEQ IDNO. 12 HBVpreS/2-21^(stearoyl)(consensus) Genotype C peptides*HBVpreS/(−11)-48^(myr)(C) natural SEQ ID NO. 4*HBVpreS/(−11)-48^(stearoyl)(C) *HBVpreS/2-48^(myr)(C) SEQ ID NO. 13*HBVpreS/2-48^(stearoyl)(C) HBVpreS/5-48^(myr)(C) truncated N- SEQ IDNO. 14 HBVpreS/5-48^(stearoyl)(C) terminal HBVpreS/9-48^(myr)(C) SEQ IDNO. 15 HBVpreS/9-48^(stearoyl)(C) HBVpreS/2-21^(myr)(C) truncated N- SEQID NO. 16 HBVpreS/2-21^(stearoyl)(C) and/or HBVpreS/5-21^(myr)(C) C- SEQID NO. 17 HBVpreS/5-21^(stearoyl)(C) terminal HBVpreS/9-21^(myr)(C) SEQID NO. 18 HBVpreS/9-21^(stearoyl)(C) HBVpreS/2-15^(myr)(C) SEQ ID NO. 19HBVpreS/2-15^(stearoyl)(C) HBVpreS/5-15^(myr)(C) SEQ ID NO. 20HBVpreS/5-15^(stearoyl)(C) HBVpreS/9-15^(myr)(C)** SEQ ID NO. 21HBVpreS/9-15^(stearoyl)(C)** HBVpreS/(−2)-20^(palm)(C) SEQ ID NO. 22Genotype D peptides HBVpreS/1-48^(myr)(D) natural SEQ ID NO. 5HBVpreS/2-48^(myr)(D) SEQ ID NO. 23 HBVpreS/2-48^(stearoyl)(D)HBVpreS/5-48^(myr)(D) truncated N- SEQ ID NO. 24HBVpreS/5-48^(stearoyl)(D) terminal HBVpreS/9-48^(myr)(D) SEQ ID NO. 25HBVpreS/9-48^(stearoyl)(D) HBVpreS/2-21^(myr)(D) N- SEQ ID NO. 26HBVpreS/2-21^(stearoyl)(D) and/or HBVpreS/5-21^(myr)(D) C- SEQ ID NO. 27HBVpreS/5-21^(stearoyl)(D) terminal HBVpreS/9-21^(myr)(D) SEQ ID NO. 28HBVpreS/9-21^(stearoyl)(D) HBVpreS/2-20^(myr)(D) SEQ ID NO. 38HBVpreS/2-20^(stearoyl)(D) HBVpreS/2-15^(myr)(D) SEQ ID NO. 29HBVpreS/2-15^(stearoyl)(D) HBVpreS/5-15^(myr)(D) SEQ ID NO. 30HBVpreS/5-15^(stearoyl)(D) HBVpreS/9-15^(myr)(D)** SEQ ID NO. 21HBVpreS/9-15^(stearoyl)(D)** ^(myr)refers to myristoylation of theN-terminus; ^(palm)refers to palmitoylation of the N-terminus;^(stearoyl)refers to stearoylation of the N-terminus; (C)refers togenotype C (with Q46K, as in SEQ ID NO. 4); (D)refers to genotype D;**minimal sequence.

TABLE 2 Preferred hydrophobic modified preS-derived peptides withchanges in the immunogenic epitopes Designation of peptide Amino acidsequence HBVpreS/2-48^(myr)-Ala^(21,23,29,30)(C) SEQ ID NO. 31HBVpreS/2-48^(stearoyl)-Ala^(21,23,29,30)(C)HBVpreS/(−11)-48^(myr)-D20A(C) SEQ ID NO. 32HBVpreS/(−11)-48^(stearoyl)-D20A(C) HBVpreS/2-48^(myr)-D20A(C) SEQ IDNO. 33 HBVpreS/2-48^(stearoyl)-D20A(C)HBVpreS/(−11)-48^(myr)-SNN(27-29)ANA(C) SEQ ID NO. 34HBVpreS/(−11)-48^(stearoyl)-SNN(27-29)ANA(C)HBVpreS/2-48^(myr)-SNN(27-29)ANA(C) SEQ ID NO. 35HBVpreS/2-48^(stearoyl)-SNN(27-29)ANA(C) HBVpreS/(−11)-48^(myr)-D20A +SEQ ID NO. 36 SNN(27-29)ANA(C) HBVpreS/(−11)-48^(stearoyl)-D20A +SNN(27-29)ANA(C) HBVpreS/2-48^(myr)-D20A + SNN(27-29)ANA(C) SEQ ID NO.37 HBVpreS/2-48^(stearoyl)-D20A + SNN(27-29)ANA(C) ^(myr)refers tomyristoylation of the N-terminus; ^(stearoyl)refers to stearoylation ofthe N-terminus; (C)refers to genotype C (with Q46K, as in SEQ ID NO. 4);

The following examples and drawings illustrate the present inventionwithout, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the HBV particle and the HBV L-, M-and S-proteins.

(A) The partially double stranded DNA is covalently associated with theviral polymerase complex, consisting of the terminal protein, (TP), thereverse transcriptase (RT) and the RNaseH. The genome is encapsulated byan icosahedral shell, built of 120 core-protein dimers. The 3 HBVsurface proteins L-, M- and S- are embedded into an ER-derived lipidbilayer. The L- and M-proteins contain the complete S-domain serving asa membrane anchor.(B) Domain structure of the 3 HBV surface proteins L, M and S.The L-protein contains the N-terminally myristoylated 107 amino acidpreS1-domain, the 55 amino acid preS2-domain and the S-domain containingthe 4 transmembrane segments (I-IV).

FIG. 2. HBV preS1 consensus sequence.

At the top: the HBV L-protein with its preS1, preS2 and S-domain isdepicted. The N-terminus is myristoylated.

The alignment below shows: the consensus sequence (Consensus) and theeight HBV genotypes (A-H) as well as the woolly monkey HBV (WMHBV) preSsequence encompassing amino acids 2-48. Note that the genotypes A, B, C,E, G and H have eleven additional amino acids at their N-termini,genotype F has 10 additional amino acid residues. At the bottom, theknown functional subdomains are shown.Please note that HBV genotype C refers to HBV genotype C Q46K.

FIG. 3. Biodistribution and liver stability of HBVpreS-derivedlipopeptides after subcutaneous application.

(A) Liver-specific accumulation of myristoylated (C₁₄) versusstearoylated (C₁₈) HBV preS/2-48 (D) peptides after subcutaneousinjection in male NMRI mice in comparison to the control Fuzeon® (T-20).Per animal approximately 2.25 μg of the ¹³¹I-labelled peptides weresubcutaneously injected. At the indicated time points animals weresacrificed and the liver specific peptide accumulation (% ID/g) wasdetermined.(B) Reversed phase HPLC of pure ¹³¹I-labelled HBVpreS/2-48^(myr)(D)(brown curve) in comparison to liver extracts (orange curve) obtained 24h post subcutaneous injection into uPA+/−/RAG-2−/−Pfp−/− mice of thesame peptide. Note that 50% of detectable activity is eluting atfraction 13. Since the hydrophobic myristoyl residue is located at theN-terminus and the iodinated Tyr-residue at the C-terminus fraction 13represents the unaltered full-length peptide.

FIG. 4. Biodistribution of HBVpreS/2-48^(myr)(D) after subcutaneousapplication in NMRI mice.

Per time point 2.25 μg of ¹³¹I-labeled HBVpreS/2-48^(myr)(D) weresubcutaneously injected into NMRI mice (N=3). 10 min, 1 h, 4 h and 24 hpost injection animals were sacrificed and the organ-specific (blood,lung, heart, spleen, liver, kidney and muscle) peptide accumulations (%ID/g) were determined. Note that most of the peptide accumulates in theliver and that the Y-axis is jolted for better resolution of thedistribution in the other organs.

FIG. 5. Biodistribution of HBVpreS/2-48^(myr)(D) after subcutaneousapplication in uPA+/−/RAG-2−/−Pfp−/− mice.

Per time point 2.25 μg of ¹³¹I-labeled HBVpreS/2-48^(myr)(D) weresubcutaneously injected (N=3). 10 min, 1 h, 4 h and 24 h post injectionanimals were sacrificed and the organ-specific (liver, spleen, kidney,blood, lung, intestine, heart, muscle and brain) peptide accumulations(% ID/g) were determined. Note that like in NMRI mice most of thepeptide accumulates in the liver.

FIG. 6. Scintigram of ¹²⁵I-labelled HBVpreS/2-48^(stearoyl)(D) aftersubcutaneous injection in a mouse.

Specific accumulation of ¹²⁵I-labelled HBVpreS/2-48^(stearoyl)(D) in theliver of mice after subcutaneous injection near the right leg; liverappears on the left side in the middle. Note that the upcoming signalnear the head is the thyroid and might represent free iodine afterde-iodination of the peptide.

FIG. 7. The liver tropism is sequence specific and requires N-terminalhydrophobic modification.

A and B. Biodistribution of a preS peptide, wherein the N-terminus isnot hydrophobic modified, compared to a randomized (“scrambled”)sequence. Note that both HBVpreS/1-48 Tyr as well as its randomized formare located to the kidney.

A: Biodistribution of HBVpreS/1-48 Tyr(D) in mice.

B: Biodistribution of HBVpreS/scrambled 1-48 Tyr(D) in mice.

C and D. Biodistribution of a hydrophobic modified preS peptide comparedto a randomized (“scrambled”) sequence. Only HBVpreS/2-48Tyr^(stearoyl)(D) is transported to the liver, wherein its randomizedform is evenly distributed.

C: Biodistribution of HBVpreS/2-48 Tyr^(stearoyl)(D) in mice.

D: Biodistribution of HBVpreS/2-48 Tyr (scrambled)^(stearoyl)(D) inmice.

FIG. 8. Biodistribution of variants of hydrophobic modified preSpeptides, wherein the variants are N- and/or C-terminal truncated aswell as point mutated.

The truncated variants, HBVpreS/5-48 D-Tyr^(stearoyl)(D), HBVpreS/2-33-DTyr^(stearoyl)(D) as well as HBVpreS/2-21 D-Tyr^(stearoyl)(D), showliver tropism, whereas the variant with a point mutation at position 12(G12E) does not.

A: Biodistribution of HBVpreS/5-48 D-Tyr^(stearoyl)(D) in mice.

B: Biodistribution of HBVpreS/2-33-D Tyr^(stearoyl)(D) in mice.

C: Biodistribution of HBVpreS/2-48(G12E) D-Tyr^(stearoyl)(D) in mice.

D: Biodistribution of HBVpreS/2-21 D-Tyr^(stearoyl)(D) in mice.

FIG. 9. HBV infection inhibition by hydrophobic modified HBVpreS-derivedpeptides of the invention.

A: HBV infection inhibition by HBVpreS/2-48^(myr)(D),HBVpreS/2-48^(myr)(C), HBVpreS/(−11)-48^(myr)(C),HBVpreS/2-48^(stearoyl)(C) and HBVpreS/(−11)-48^(stearoyl)(C).

B: HBV infection inhibition by HBVpreS/2-48^(stearoyl)(D),HBVpreS/2-15^(stearoyl)(D), HBVpreS/2-21^(stearoyl)(D),HBVpreS/2-26^(stearoyl)(D) and HBVpreS/2-33^(stearoyl)(D).

HepaRG cells were infected either in absence (0 nM) or in the presenceof 1, 5, 25, 100 and 1000 nM of the respective hydrophobic modifiedHBVpreS-derived peptides of the invention. The infectious inoculum (HBVof genotype D) and the peptides were incubated overnight. After washing,cells were maintained for another 12 days to allow viral geneexpression. Cell culture supernatants from day 8-12 were collected andanalyzed for secreted HBSAg using a quantitative commercially availableELISA. HBsAg values from the respective uncompleted infection were setto 100% and the degree of infection inhibition is given in % of theuncompleted infection.Wherein (C) or genotype C refers to HBV genotype C Q46K.

FIG. 10. Binding of the hydrophobic modified HBVpreS-derived peptides ofthe invention to primary human hepatocytes (PHH) is myristoyl-dependentand sequence specific

Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

Primary human hepatocytes (PHH) were incubated with or without 400 nMpeptide for 4 hours:

A without peptide

B with wt myr-FITC

C with mut myr-FITC

D with wt myr(−)-FITC

Bright (green) dots show autofluorescence of the cells (A, C and D),whereas only in B binding of the peptide to the cells can be seen.

wt myr-FITC refers to HBVpreS/2-48^(myr)(C)-FITC with lysine (K) at theC-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC)

mut myr-FITC HBVpreS/2-48^(myr)(C)-FITC with two amino acidsubstitutions in positions L11 and F13, wherein the amino acids weresubstituted with the respective D amino acid residues (D-Leu and D-Phe)[SEQ ID NO. 40], labelled with a fluorophor (FITC)

wt myr(−)-FITC refers to HBVpreS/2-48(C)-FITC without myristoylation[SEQ ID NO. 39], labelled with a fluorophor (FITC)

The fluorophor FITC was attached to the sidechain (NFL; group) of theadditional Lys.

FIG. 11. Binding of the hydrophobic modified HBVpreS-derived peptides ofthe invention is not restricted to HBV-susceptible hepatocytes.

Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

Cells were incubated with or without 400 nM peptide for 4 hours at 37°C.:

A (upper panel) Differentiated HepaRG cells

B (lower panel) Primary mouse hepatocytes (PMH)

Only wt myr-FITC binds to the cells. Bright (green) dots showautofluorescence of the cells

wt myr-FITC refers to HBVpreS/2-48^(myr)(C)-FITC with lysine (K) at theC-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC) attachedto the sidechain (NH₂ group) of the additional Lys

mut myr-FITC HBVpreS/2-48^(myr)(C)-FITC with two amino acidsubstitutions in positions L11 and F13, wherein the amino acids weresubstituted with the respective D amino acid residues (D-Leu and D-Phe)[SEQ ID NO. 40], labelled with a fluorophor (FITC)

wt myr(−)-FITC refers to HBVpreS/2-48(C)-FITC without myristoylation[SEQ ID NO. 39], labelled with a fluorophor (FITC)

The fluorophor FITC was attached to the sidechain (NH₂ group) of theadditional Lys.

FIG. 12. Binding of the hydrophobic modified HBVpreS-derived peptides ofthe present invention depends on the differentiation state of the cells.The peptides only bind to differentiated hepatocytes.

Immunofluorescence, showing confocal images Blue: dapi; Green: FITC.

Cells were incubated with or without 400 nM peptide for 4 hours at 37°C.:

A (upper panel) Comparing differentiated and undifferentiated HepaRGcells

B (lower panel) Comparing differentiated and de-differentiated Primarymouse hepatocytes (PMH)

wt myr-FITC binds only to differentiated cells. Bright (green) dots showautofluorescence of the cells

wt myr-FITC refers to HBVpreS/2-48^(myr)(C)-FITC with lysine (K) at theC-terminus [SEQ ID NO. 39], labelled with a fluorophor (FITC)

The fluorophor FITC was attached to the sidechain (NH₂ group) of theadditional Lys.

FIG. 13. FACS analysis of the binding of a hydrophobic modifiedHBVpreS-derived peptide of the present invention to primary mousehepatocytes (PMH).

A showing the protocol of the FACS analysis

B showing the results of the FACS analysis (in comparison to theimmunofluorescence results). Only wt myr-FITC binds to the cells.

wt myr-FITC refers to HBVpreS/2-48^(myr)(C)-FITC with lysine (K) at theC-terminus [SEQ ID NO. 41], labelled with a fluorophor (FITC)

mut myr-FITC HBVpreS/2-48^(myr)(C)-FITC with two amino acidsubstitutions in positions L11 and F13, wherein the amino acids weresubstituted with the respective D amino acid residues (D-Leu and D-Phe)[SEQ ID NO. 42], labelled with a fluorophor (FITC)

The fluorophor FITC was attached to the sidechain (NH₂ group) of theadditional Lys.

FIG. 14. Fusion constructs of HBVpreS-derived peptides with mouseinterferon alpha.

FIG. 15. Purification and activity test ofHBVpreS/2-20^(myr)(D)-IFNalpha

Purification of the fusion protein HBVpreS/2-20^(myr)(D)-mouseInterferon alpha 2 from the supernatant of insect cells, which wereinfected with a recombinant baculovirus.

A First chromatography step: chromatographic purification via His-Tag,using Ni-agarose, because the HBVpreS/2-20^(myr)(D)-IFNalpha constructcarries a C-terminal His-tag.

B Second chromatography step: gel filtration chromatography (S75)

C The elution fractions of the first chromatography step were tested forIFN.

See also Examples.

For the amino acid sequence of HBVpreS/2-20(D) see SEQ ID NO. 38.

EXAMPLES

Synthesis of the Hydrophobic Modified preS-Derived Peptides

The synthesis was carried out as described e.g. in (10).

Biodistribution of the Hydrophobic Modified preS-Derived Peptides

The biodistribution of the hydrophobic modified preS-derived peptideswas studied in male NMRI mice. All experiments were performed incompliance with German laws. The peptides, containing an additionalTyr-residue at the C-terminal end were labelled with ¹³¹I (AmershamBiosciences, Freiburg, Germany) by the chloramine-T method and purifiedby HPLC. The labelled peptides were subcutaneously administered byinjection of a solution in 50% DMSO. At selected times mice weresacrificed and the radioactivity contained in the blood, heart, lung,spleen, liver, kidney, muscle and brain was measured in a γ-counter(Canberra Packard, Rüsselsheim, Germany) and expressed as a percentageof injected dose per gram of tissue (% ID/g).

Stability Assessment of the Hydrophobic Modified preS-Derived Peptidesafter Extraction from the Liver

To determine the peptide stability in the liver 131I labelledHBVpreS/2-48^(myr) (D) was extracted from one liver lobe 24 h postsubcutaneous injection. To that aim, 1 ml water per gram frozen livertissue was added to the sample. After homogenization an equal volume ofacetonitrile was added and the homogenization was repeated. Aftercentrifugation (2×10 min at 4000×g) this solution was separated on areverse phase HPLC column and the radioactivity of each fraction wasquantified in a gamma counter.

Cell Lines and Primary Cell Cultures.

HepaRG cells were grown in William's E medium supplemented with 10%fetal calf serum (FCS), 100 units/ml penicillin, 100 μg/ml streptomycin,5 μg/ml insulin and 5×10⁻⁵ M hydrocortisone hemisuccinate (10). Cellswere passaged 1/5 every two weeks by trypsination. Two to three weeksbefore infection cell differentiation was induced by adding 2% DMSO intothe maintenance medium. The medium was exchanged every 2-3 days.

Infection Competition Assays.

As an infectious inoculum, a 50-fold concentrated culture supernatant ofHepG2 clone 2.2.15 (11) cells was used, because of an unlimited supplyand a constant quality. It was prepared from freshly collectedsupernatants by precipitating viral particles in the presence of 6%polyethylene glycol (PEG) 8000. The pellet was resuspended in phosphatebuffered saline (PBS) containing 25% FCS. Aliquots were stored at −80°C. Differentiated HepaRG cells or PHH were incubated with theconcentrated infectious source, 10-fold diluted in culture mediumsupplemented with 4% PEG 8000 (Sigma), for 20 h at 37° C. At the end ofthe incubation, cells were washed three times with the culture mediumand maintained in the presence of 2% DMSO and 5×10⁻⁵ M hydrocortisonehemisuccinate and harvested at indicated times.

Competition experiments were performed in 12-well plates. Approximately1×10⁶ cells were first pre-incubated for 30 min with chemicallysynthesized HBV derived peptides followed by a co-incubation of cellswith peptide and virus for 20 h. All competition series were performedat least twice and the results of one representative experiment areshown in each case (see FIGS. 3 to 7).

HepaRG cells were infected either in absence (0 nM) or in the presenceof 1, 5, 25, 100 and 2000 nM of the respective peptides of theinvention. The infectious inoculum (HBV of genotype D) and the peptideswere incubated overnight. After washing, cells were maintained foranother 12 days to allow viral gene expression. Cell culturesupernatants from day 8-12 were collected and analyzed for secretedHBSAg using a quantitative commercially available ELISA. HBsAg valuesfrom the respective uncompleted infection were set to 100% and thedegree of infection inhibition is given in % of the uncompletedinfection.

Immunofluorescence Experiments/Microscopy

Primary hepatocytes grown on cover slips were incubated with therespective peptide at 200 nM in serum-free cell culture medium. After 1hour incubation at 37° C. cells were fixed and nuclei were stained withDAPI. Fluorescence microscopy was performed on a Spinning Disk ConfocalMicroscope at 600× magnification.

FACS Analysis

Cryo-preserved primary hepatocytes were thawed and washed withserum-free medium. In each reaction 4×10⁵ cells/ml were incubated withthe respective peptide at a concentration of 200 nM in serum-free cellculture medium. The staining was performed for 30 minutes at roomtemperature with frequent mixing. Subsequently, cells were washedextensively and resuspended in PBS to proceed with the FACS analysis.

Results are shown in the Figures.

Fusion Constructs of HBVpreS1-Peptides with Mouse InterferonAlpha-Production, Purification and Activity Test

HBVpreS1-peptide-mouse interferon alpha fusion proteins (see FIG. 14)were expressed in Hi5-insect cells using the baculovirus expressionsystem. The fusion proteins secreted in the cell supernatants wereharvested on day 5 post infection and purified in a first step by anaffinity chromatography for the His-tag fused C-terminally to thepreS1-interferon. The activity of the preS1-interferon proteins in theelution fractions was measured by their ability to inhibit Newcastledisease virus mediated cell death. The elution fractions containingfunctional preS1-interferon proteins were further purified by gelfiltration chromatography on a S75-sepharose column with a 1M urea-PBSbuffer (see FIG. 15).

The features disclosed in the foregoing description, in the claimsand/or in the accompanying drawings may, both separately and in anycombination thereof, be material for realizing the invention in diverseforms thereof.

REFERENCES

-   1. Seeger, C. & Mason, W. S. Hepatitis B virus biology. Microbiol.    Mol Biol Rev 64, 51-68 (2000).-   2. Nassal, M. Hepatitis B virus morphogenesis. Curr Top Microbiol    Immunol 214, 297-337 (1996).-   3. Gripon, P., Le Seyec, J., Rumin, S. & Guguen-Guillouzo, C.    Myristylation of the hepatitis B virus large surface protein is    essential for viral infectivity. Virology 213, 292-299 (1995).-   4. Le Seyec, J., Chouteau, P., Cannie, I., Guguen-Guillouzo, C. &    Gripon, P. Infection process of the hepatitis B virus depends on the    presence of a defined sequence in the preS1 domain. J Virol 73,    2052-2057 (1999).-   5. Juliano R L (1988) Factors affecting the clearance kinetics and    tissue distribution of liposomes, microspheres, and emulsions. Adv    Drug Deliv Rev 2:31-54.-   6. Hashida M and Takakura Y (1994) Pharmacokinetics in design of    polymeric drug delivery systems. J Control Release 31: 163-171.-   7. Lu, X. M., Fischman, A. J., Jyawook, S. L., Hendricks, K.,    Tompkins, R. G. and Yarmush, M. L. (1994) Antisense DNA delivery in    vivo: liver targeting by receptor-mediated uptake. J. Nucl. Med. 35,    269-275.-   8. Freireich, E. J., Gehan, E. A., Rall, D. P., Schmidt, L. H. &    Skipper, H. E. Quantitative comparison of toxicity of anticancer    agents in mouse, rat, hamster, dog, monkey, and man. Cancer    Chemother Rep 50, 219-244 (1966).-   9. Erion, M. D. Prodrugs for liver-targeted drug delivery. 541-572.    Biotechnology: Pharmaceutical Aspects. Volume V, Part II, Prodrugs.    Springer New York, 2007.-   10. Gripon, P., Cannie, I. & Urban, S. Efficient inhibition of    hepatitis B virus infection by acylated peptides derived from the    large viral surface protein. J Virol 79, 1613-1622 (2005).-   11. Glebe D, Urban S, Knoop E V, Cag N, Krass P, Grun S, Bulavaite    A, Sasnauskas K, Gerlich W H. Mapping of the hepatitis B virus    attachment site by use of infection-inhibiting preS1 lipopeptides    and tupaia hepatocytes. Gastroenterology 129, 234-245 (2005).

The invention claimed is:
 1. A hydrophobic modified preS-derived peptideof hepatitis B virus (HBV) of the formula[H_(m)-P-R_(n)]A₀ wherein P is a preS-derived peptide consisting of: a)an N-terminally and/or C-terminally truncated variant of HBV preSconsensus sequence as shown in SEQ ID NO: 1, wherein the truncatedvariant consists of a minimum of 10 and a maximum of 46 amino acids; orb) a truncated variant of HBV preS consensus sequence as shown in SEQ IDNO: 1, wherein the truncated variant consists of a minimum of 7 and amaximum of 46 amino acids, and wherein the truncated variant comprisesthe sequence of SEQ ID NO: 21; wherein, H is an acylation of thepreS-derived peptide P that is N-terminal of P, m is at least 1; R is aC-terminal modification of said preS-derived peptide P, which is amoiety that protects from degradation, selected from amides, albumin,natural polymers, and synthetic polymers; n is 0 or at least 1; A is ananchor group, selected from ester, ether, disulfide, amide, thiol, andthioester; and ₀ is 0 or at least 1; wherein said hydrophobic modifiedpeptide further comprises at least one compound selected from drugs;prodrugs; labels; recombinant viruses or derivatives thereof; carriersor depots for a drug, prodrugs or labels; and immunogenic epitopes;wherein the label is a contrast agent; and wherein the compound and thehydrophobic peptide form a conjugate.
 2. The hydrophobic modifiedpreS-derived peptide according to claim 1, wherein the acylation isselected from acylation with myristoyl (C 14), palmitoyl (C 16) orstearoyl (C 18).
 3. The hydrophobic modified preS-derived peptideaccording to claim 1, wherein H and/or R is linked to P via a linker orspacer.
 4. The hydrophobic modified preS-derived peptide according toclaim 1, wherein P consists of N-terminally and/or C-terminallytruncated variants of a minimum of 20 and a maximum of 46 consecutiveamino acids of SEQ ID NO:
 1. 5. The hydrophobic modified preS-derivedpeptide according to claim 1, wherein P consists of the N-terminallyand/or C-terminally truncated variants of a maximum of 46 consecutiveamino acids of SEQ ID NO: 1 and comprises an amino acid sequenceselected from amino acid residues 2 to 21, residues 2 to 20, or residues9 to 15 of the HBV preS consensus sequence as shown in SEQ ID NO:
 1. 6.The hydrophobic modified preS-derived peptide according to claim 1,wherein P does not comprise amino acid substitutions and/or deletions atresidues 9 to 15 of SEQ ID NO:
 1. 7. The hydrophobic modifiedpreS-derived peptide according to claim 1, wherein P consists of theN-terminally and/or C-terminally truncated variants of a maximum of 46consecutive amino acids of SEQ ID NO: 1 and comprises an amino acidsequence of SEQ ID NO:
 38. 8. The hydrophobic modified preS-derivedpeptide according to claim 1, wherein P comprises an amino acid sequenceselected from SEQ ID NOs: 31 to 37 and variants thereof.
 9. Thehydrophobic modified preS-derived peptide according to claim 1, whereinP comprises an amino acid sequence selected from SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 26, SEQID NO: 31, and SEQ ID NO:
 38. 10. The hydrophobic modified preS-derivedpeptide according to claim 1, wherein the acylation is selected fromacylation with stearoyl (C 18) or acylation with myristoyl (C 14).
 11. Apharmaceutical composition comprising; at least one hydrophobic modifiedpreS-derived peptide of HBV according to claim 1, and at least onecompound to be specifically delivered to the liver; wherein the compoundmay be conjugated to the peptide; and, optionally, a pharmaceuticallyacceptable carrier and/or excipient.
 12. A method for the identificationof a HBV receptor, comprising the use of at least one hydrophobicmodified preS-derived peptide of HBV according to claim 1, and at leastone compound to be specifically delivered to the liver, wherein thecompound is conjugated to the peptide; and, optionally, apharmaceutically acceptable carrier and/or excipient.
 13. Thehydrophobic modified preS-derived peptide according to claim 1, whereinthe compound is selected from interferons; viral reverse transcriptaseinhibitors; viral RNA polymerase inhibitors; viral core assemblyinhibitors or viral nucleocapsid inhibitors; kinase inhibitors;nucleoside analogues; protease inhibitors; proteasom inhibitors;antibodies or fragments thereof; siRNA or precursors thereof;farnesylation inhibitors; alcohol dehydrogenase (ADH) or an activatorthereof; cholesterol biosynthesis inhibitors; inhibitors of the liverstage of a virus or a non-viral pathogen; and antibiotics.
 14. Thehydrophobic modified preS-derived peptide according to claim 13, whereinthe drug is in the form of a prodrug.
 15. The hydrophobic modifiedpreS-derived peptide according to claim 13, wherein the compound isinterferon and is attached to a peptide consisting of the N-terminallyand/or C-terminally truncated variants of a maximum of 46 consecutiveamino acids of SEQ ID NO: 1 and that comprises an amino acid sequence ofSEQ ID NO:
 38. 16. The hydrophobic modified preS-derived peptideaccording to claim 15, wherein H is a hydrophobic modification byacylation.
 17. The hydrophobic modified preS-derived peptide accordingto claim 1, wherein the conjugate is formed by covalent attachment or bycomplex formation.
 18. The hydrophobic modified preS-derived peptideaccording to claim 17, wherein the conjugate is formed by covalentlyattaching a compound to an anchor group A.
 19. The hydrophobic modifiedpreS-derived peptide according to claim 18, wherein the anchor group Afurther comprises a spacer or linker.
 20. The hydrophobic modifiedpreS-derived peptide according to claim 19, wherein the spacer or linkercomprises a recognition site, which is recognized by a liver protein.